In a development that holds
ramifications for gene therapy and infection-fighting drugs, a research
collaboration led by Carlos Bustamante of Berkeley Lab's Physical Biosciences
Division has discovered the mechanism by which at least some viruses infect
the cells of other organisms with their DNA. The mechanism involves one
of the most powerful biomolecular motors ever observed.

CARLOS BUSTAMANTE

"This motor pulls with
about 57 to 60 piconewtons of force, which scaled up to human dimensions
would be enough to lift six aircraft carriers," says Bustamante,
a biophysicist who holds a joint appointment with Berkeley Lab and UC
Berkeley, and who is also a Howard Hughes Medical Institute investigator.

Biomolecular motors are proteins
that undergo shape changes to generate force or torque. Acting like tiny
engines, biomolecular motors come in a wide assortment and perform a broad
range of tasks, many involving movement and transportation. One such task
is the packing of the coiled lengths of DNA into the protective external
shell or "capsid" of a number of viruses including those that
cause herpes, chicken pox, and shingles. The biomolecular motor that Bustamante
and his colleagues observed is the portal motor for the bacteriophage
f29 (phi-29), a virus that infects
and destroys soil bacteria and is considered an excellent model system
for studying viral assembly.

"The
portal motor for bacteriophage f29
compresses the DNA 6,000 times its normal volume," says Bustamante.
"This generates an internal pressure of about 60 atmospheres, which
is about ten times that in a champagne bottle."

THE BIOMOLECULAR PORTAL MOTOR OF
BACTERIOPHAGE PHI-29 (YELLOW) COMPRESSES COILED LENGTHS OF DNA INTO
THE VIRAL CAPSID TO 6,000 TIMES ITS NORMAL VOLUME, CREATING PRESSURES
TEN TIMES AS POWERFUL AS THOSE INSIDE A CHAMPAGNE BOTTLE.

Bustamante and his colleagues
propose that just as the internal pressure in a champagne bottle will
pop a champagne cork, so too does the even greater internal pressure inside
the bacteriophage's capsid forcibly inject the viral DNA into an attacked
cell. Viruses cannot "live" or reproduce without getting inside
a living cell, whether it's a plant, animal, or a bacterium; in the case
of f29, the bacteriophage attaches itself to
and introduces its DNA into a soil bacterium, which, unlike the virus,
can reproduce on its own. The viral DNA takes over the bacterium's reproductive
programming and instructs it to reproduce copies of the virus instead.
So many copies of the virus are replicated that the bacterium ultimately
bursts open, unleashing a mass of new viruses ready to infect other bacteria.

"Understanding how this
DNA packing process works could help us design better drugs to interfere
with the packing part of the infection cycle of the virus and perhaps
halt infection," Bustamante says. "It might also be used in
gene therapy as a means of transporting new genetic material into cells."

The results of this research
were reported in the October 18 issue of the journal Nature. Coauthoring
the paper with Bustamante were Doug Smith, now with UC San Diego; Sander
Tans, now at the Institute for Atomic and Molecular Physics in Amsterdam;
UC Berkeley's Steven Smith; and Shelley Grimes and Dwight Anderson of
the University of Minnesota.

"I would like to emphasize
the close collaboration between my laboratory and that of Dwight Anderson
that made possible this work," says Bustamante. "It was only
because of the excellent complementary expertise of the two laboratories
that this phase of the work was successfully completed."

To measure the strength of bacteriophage
f29's portal motor, the research team used
a unique force-measuring "optical tweezers" setup that was built
in Bustamante's laboratory by Steve Smith. Working with capsids that were
only partially packed with DNA before the packing process was stalled,
the researchers tethered the unpacked end of the DNA and the capsid into
which it was being packed between a pair of micron-sized polystyrene beads.

While the capsid-attached bead
was held in place by a pipette, the DNA-attached bead was captured by
the optical tweezers  a laser beam that can be used to grasp and move
the beads.

In the presence of adenosine
triphosphate (ATP), the fuel that powers many biomolecular motors, the
researchers were able to observe virus DNA packing activity in real-time
and measure the force being applied by bacteriophage f-29's
biomolecular motor. This enabled them to calculate the total amount of
work involved, the total internal pressure on the DNA, and the amount
of potential energy available for ejecting the DNA out of the capsid and
into a bacterium during infection.

"The 57 to 60 piconewtons
we calculated as the maximum pull exerted by this motor is an enormous
force," Bustamante said. "The question is, then, what happens
to all the work done on the DNA during packing? We claim the energy gets
stored up inside the head of the bacteriophage and becomes available to
initiate rapid injection of the DNA during the next infection phase."

Bustamante and his colleagues
next want to answer some fundamental questions about bacteriophage f-29's portal motor, such as whether it is a
new class of rotary biomolecular motor, one that can couple rotation to
DNA translocation.

Says UCSD's Doug Smith, "The
motor, consisting of a 10-nanometer diameter ring of RNA molecules sandwiched
between two protein rings, is very intriguing and different from other
motors that have been studied. We suspect that rotation of the rings may
pull the double helical DNA through the portal similar to the way a rotating
nut can pull on a bolt."

This work was funded by the
Department of Energy, the National Institutes of Health, and the National
Science Foundation.